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Nucleic Acids Research, 1985, Vol. 13, No. 15 5457-5469
© 1985

Articles

Separation of complementry strands of plasmid DNA using the biotin-avidin system and its application to heteroduplex formation and RNA/DNA hybridizations in electron microscopy

Hajo Delius, Harm van Heerikhuizen1, Jill Clarke and Barbara Koller

European Molecular Biology Laboratory Postfach 10.2209, 6900 Heidelberg, FRG 1Free University, Biochemical Laboratory de Boelelaan 1083, 1081 HV Amsterdam, The Netherlands

Received May 31, 1985. Accepted July 15, 1985.

A method for the separation of complementary strands with the help of the biotin-avidin system is described. Restriction fragments were terminally labeled at both ends with biotinylated nucleotides. The DNA was cut by a second restriction enzyme, and the fragments were bound to an avidin agarose column. The non-biotinylated strands were eluted with 0.1 M NaOH, and the biotin-labeled strands were subsequently released from the column by elution with 50% guanidine isothiocyanate/formamide. Contamination of the separated strands by complementary single strands was less than 4%.- Separated linear single strands of the vector pEMBL were prepared. On annealing with recombinant circular DNA a substitution loop is formed which provides position and orientation markers for the unambiguous electron microscopic analysis of heteroduplexes or hybrids formed with the inserted sequences. - The terminal biotin label was visualized by complex formation with a streptavidin-ferritin conjugate.


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